Comey declined to reveal the name of the man, who top U.K. officials previously have said appeared to be British. Comey told ABC News the FBI and international partners were able to identity him.
In several ISIS videos in which Western hostages appear to be beheaded, an armed militant -- possibly the same man every time -- stands beside them and delivers statements against Western actions against ISIS in Iraq. Then he takes his knife to their throats.
American journalists James Foley and Steven Sotloff, and British aid worker David Haines were all killed in a similarly public, brutal fashion. The life of another British citizen, Alan Henning, was threatened in the last video.
ISIS has been wreaking havoc as it captures large swaths of territory in Iraq and Syria, and the American military now has the group in its sights amid a sustained, U.S.-involved bombing campaign in Syria. In addition the group's capabilities in that region, U.S. officials have expressed concern over the group's "very slick media campaign" pushing out propaganda online and using social media to encourage people around the world to launch attacks in their homelands.
Part of that media campaign is a recent series of propaganda videos, including two showing the beheadings of Foley and Sotloff.
The latest video released by ISIS earlier this week, titled "Flames of War," is narrated by another man speaking perfect English, but "there is no doubt [he] is speaking North American-accented English," Comey said. And investigating him further "is a big focus of ours," Comey said.
Comey also spoke today about the Khorasan Group, another target of U.S. airstrikes this week in Syria. The little-known group was "nearing the execution phase for an attack in Europe or the homeland," U.S. officials have said. And the group was working to produce "creative" new designs for bombs that could be smuggled onto U.S.- or Europe-bound flights, sources have said.
"That group was at the top of my list of things that I worry about ... [because] it is a collection of very bad and experienced terrorists, operating in a safe haven, into which we don't have complete visibility," Comey said today.
Due to that "limited visibility," it's hard to say whether their attack would come "tomorrow, three weeks from now or three months from now," he said. "But it's the kind of threat you have to operate under the assumption that it is tomorrow."
In a one-on-one conversation with ABC News' Pierre Thomas just hours after his pending resignation was announced, Attorney General Eric Holder said there's no doubt Khorasan is "a real threat."
"Trust me, those of us who have been in the situation room, we have been following them. ... [And] we know what their capabilities are," Holder said.
As for how effective the airstrikes were against Khorasan, Holder said, "Until we know that their capabilities have been very, very degraded, that key people have been eliminated," authorities have to assume that the group remains a threat.
Comey echoed that sentiment in his briefing with reporters, saying that because the strikes against Khorasan involved a "large group of people," he is "operating under the assumption" that "at least some portion of the group is alive."
Holder would not confirm that the United States believes it has identified the ISIS operative seen in the beheading videos, saying only, "We're still working with our British counterparts to determine exactly who that person is."
At least two other Americans are believed to be held by ISIS, one a 26-year-old woman. Speaking to reporters on Air Force One, White House Deputy National Security Adviser Ben Rhodes told reporters today the government "continue[s] to focus on whatever resources we can bring to bear to try and secure their release."
"This is something we work very actively," Rhodes said.
Last month senior U.S. officials revealed that this summer -- before the first American was killed -- an American special operations team attempted to rescue ISIS hostages outside Raqqa, Syria. The mission was "flawless," as Defense Secretary Chuck Hagel said, except that there were no hostages at the location.
ABC News’ Lee Ferran contributed to this report.